Analytical biochemistry system with robotically carried bioarray

ABSTRACT

An analytical biochemistry system featuring a substrate with reactants immobilized thereon at fixed, known locations, a holder supporting the substrate and a manipulator for transporting the holder to a fixed sample and to an inspection station. The reactants are binding agents for a target biomolecule in a sample which forms a bound substance having a detectable characteristic. The holder may be a standard pipettor, optionally carried by a robot arm or hand as the manipulator to contact the sample for detection of the presence of target biomolecules within the sample. In one embodiment, the holder is a pipette tip within which the substrate is housed, or it may be a pipette adapter which bears the substrate and fits within the sample wells of a standard microtiter plate. After appropriate incubation, the substrate and holder may be moved from contact with the sample, and the substrate may be exposed to any necessary development steps before being moved by the manipulator to a location for probing, such as by a beam. Probing of the substrate is performed to identify binding or complexing of target biomolecules of the sample with the reactants immobilized on the substrate, and may be accomplished via fluorescence detection, light scattering, autoradiography, or some other detection method.

TECHNICAL FIELD

[0001] This invention relates to a system and methods for detecting thepresence of target biomolecules within samples with robotic assistancefor a sample holder carrying an array of reactants.

BACKGROUND ART

[0002] Assays for the detection of target biomolecules within a sample,especially of multiple target biomolecules within a sample, are oftenperformed by applying a volume of the sample to a test slide, membrane,or other substrate having immobilized reactants which may interact withthe target or targets to form detectable complexes. These immobilizedreactants are usually disposed at fixed locations, with samples broughtto these locations. U.S. Pat. No. 5,139,743, for example, discloses abiochemical analysis apparatus wherein an applicator takes up a liquidsample and applies the sample to a fixed position test film for chemicalanalysis of the sample.

[0003] Sometimes complexes of target biomolecules and reactants arevisually detectable directly after an appropriate incubation period forthe sample and reactants, or after numerous development steps whereindevelopment chemicals, such as fluorescent dye-conjugated molecules, areallowed to interact with the complexes. For example, the detectionmechanism in U.S. Pat. No. 5,296,194 involves optically detecting acolor change in a blood drop applied to a test slide.

[0004] U.S. Pat. No. 4,877,745 discloses methods for preparingimmobilized reagents and applying samples to immobilized reagents. Inparticular, this patent discloses dispensing precisely controlledvolumes of droplets onto a medium at precisely controlled locations, toform arrays of immobilized reagents by a jet head. An x-y plotter may bemodified to carry a jet head so that reagent may be dispensed over anarea.

[0005] Robotic laboratory workstations, such as the Biomek 1000 and 2000of Beckman Instruments, Inc. have been developed for automaticallycarrying out assays involving multiple reactants and multiple samples.Typically such workstations are designed to deliver robotically precisevolumes of reactants to a number of different samples located at knownareas within the workstation. Alternatively, workstations canrobotically move samples to reagents.

[0006] U.S. Pat. No. 5,171,537 to Wainwright et al. teaches activatedimmunodiagnostic pipette tips. The pipette tip houses a sphericalelement which is coated with a single ligand having affinity for atarget molecule of a sample. With this device, the test element may bebrought to contact the sample, as by aspirating the sample into thepipette tip. These pipette tips are limited in their sample throughputbecause they house only a single ligand reagent and thus preclude thedetection of multiple analytes within a sample.

[0007] A class of devices known as optical biosensors, characterized byimmobilized assay species within a supporter and a light collectiondevice coupled to an optical waveguide, is also known. Opticalbiosensors may be used for detecting and quantifying the presence ofspecific species in test fluid samples, such as in clinical diagnosticreactions. For example, U.S. Pat. No. 4,857,273 discloses an opticalbiosensor for immunoassays and certain other reactions. Other examples,involving use of an optical fiber, are U.S. Pat. No. 5,143,066 and U.S.Pat. No. 5,401,469.

[0008] It is an object of the present invention to provide apparatus andmethods for rapidly and automatically determining the presence ofmultiple target biomolecules in a single sample. It is another object ofthe present invention to provide analytical methods which requireminimal sample volume and a minimal number of liquid transfers. It is afurther object of the present invention to provide a device and systemfor rapid assessment of samples for target biomolecules which is readilyadaptable to a variety of chemical and other detection schemes.

DISCLOSURE OF THE INVENTION

[0009] The present invention achieves the above objects by providing ananalytical biochemistry system for automated analysis of samples for thepresence of target biomolecules. The system includes a solid substratewhich is supported by a holder and carried by a manipulator, such as arobotic arm. Immobilized on the solid substrate surface at discrete,site-specific locations are reactants in an array which are capable ofbinding with target biomolecules in specific binding reactions to formimmobilized biomolecule complexes. Such an array is termed a “bioarray”.The presence of target biomolecules in the sample is determined bydetecting immobilized biomolecule complexes on the bioarray with somekind of probe, e.g. a fluorescence detector. In operation, themanipulator moves the bioarray to contact the substrate surface with avolume of sample. Then the manipulator moves the contacted bioarray to adetection station to detect the absence or presence of immobilizedbiomolecule complexes. In alternative embodiments the bioarray isstationary and a sample manipulator moves samples to the holder.

[0010] In the preferred embodiment, the bioarray is mobile, beingcarried by a manipulator. A detection station is located near the sampleto probe the substrate after interaction between the reactants andsample or samples has occurred.

[0011] Distinct reactants specific to different target biomolecules areimmobilized on a preferably flat, non-porous substrate. These reactantsform a plurality of active sites on the substrate at known locations.The substrate may be a planar strip with linearly-arranged reactantsforming separable spots or bands, or may be a planar sheet having anarea-wide arrangement of reactants, forming spots or dots in atwo-dimensional array, or may be a fiber or rod with substrate disposedin a manner similar to a strip.

[0012] The holder supports the bioarray and is carried by themanipulator which transports the substrate to the location of the fixedsample, and then to the location of the detection assembly. As stated,the substrate could be fixed and the sample transported. One example ofa holder is a pipette or a pipette tip, within which a bioarray isaffixed. The sample is drawn up into the pipette tip, as with aspirationfrom a bulb or vacuum pump, or withdrawal of a plunger. The sample isthus placed in contact with the substrate, allowing any target moleculeswhich may be present within the sample to interact with the appropriatereactive sites on the substrate. After the appropriate incubation orreaction period, the sample may be removed from the pipette tip, as byair pressure or positive displacement with a plunger.

[0013] Another example of a useful holder is a pipette adapterresembling a truncated pipette tip and having a bracket or a flatsurface for supporting the substrate. The pipette adapter may be placeddirectly into a sample, such as in a well of a microtiter plate or in avial, in order to provide contact of the holder and the sample. Thepipette adapter and accompanying substrate are then removed from thesample to a detector station. The various holders of the presentinvention may be adaptations of standard pipetting tools. The holdersalso are designed to require minimal sample volumes and to allow opticalinspection of the substrate with minimal interference by the holder.

[0014] The method for detecting target biomolecules within a sampleincludes the steps of treating a substrate with a plurality of distinctreactants to form reagents immobilized on the substrate at fixed, knownpositions defining an array, i.e. a bioarray. The reactants are selectedto bind one or more target molecules to form a complex having adetectable and identifiable characteristic, such as a fluorescentsignature. The bioarray is supported in the holder. In turn, the holderhas a shape which can be picked up by a manipulator which moves thesubstrate for contact with the fixed sample, and then removes andpossibly rinses the substrate at another location to remove unboundbiomolecules. Then the manipulator moves the substrate to a probingstation, such as an optical inspection location for probing the activesites of the substrate with a beam for determining complementation ofthe target biomolecules by detecting the optically detectablecharacteristic.

[0015] Inspection may include detection of fluorescence, lightscattering, absorbance, reflectance, chemiluminescence, radioactiveemission, conductivity or electronic property. Depending on the natureof the substrate, detection of transmitted light is also possible. Priorto probing, intermediary steps to enhance visualization or realizationof complementation, such as treatment with development chemicals,fluorescent dyes, etc. may be desired. Optical inspection of thesubstrate within the pipette tip is possible by use of an opticalsurface on the pipette tip. Optical inspection on the pipette adapter isunencumbered.

[0016] A manipulator in the form of a robotic arm gripping the pipettetip or pipette adapter type of substrate holder may place the bioarrayin contact with the sample, and subsequently transfer the substrate to adetection assembly. Multiple sample transfers are thus eliminated. Acomputer controlling the robotic arm movement, the incubation times, andproviding further analysis or display of detected signals from thesubstrate is preferred. An automated instrument includes a detectionassembly, which in one embodiment includes a laser source providing anexcitation beam to impinge upon the active sites of the substrate, alight collector for gathering signals emitted from the substrate, and adetector, such as a photomultiplier tube or CCD array. Alternatively, itmay have multiple detection assemblies, depending on the requirements ofthe sample and the substrate chemistries. Relative movement of anexcitation beam and the bioarray may be provided by the robotic armholding the substrate or by scanning optics, such as a galvo mirror,within the excitation path of the detection assembly.

[0017] A substrate intended for use in the present invention may be anoligonucleotide array, a peptide array, or an immunochemical array,among others, and may be created on a separate member, such as a smallslide, and affixed to the holder, or it may be created directly on theholder. Creation of the bioarray may be via biopolymer synthesis on asolid phase member or deposition of reactants, e.g. by movable nozzles,such as the type used for ink jet printing, or by some other method. Thereactants may be affixed to the member via specific or non-specificcovalent linkages, physical adsorption, or some other form of adhesion.The interaction or complexing of the target biomolecules and theimmobilized reactants may be by affinity linkages, ionic linkages,adsorption, or some other reasonably secure manner.

[0018] The present invention provides a simple, highly adaptable methodand apparatus for quickly and easily assessing samples for the presenceof biomolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a perspective view of an automated instrument forperforming the target biomolecule detection in accord with the system ofthe present invention.

[0020]FIG. 2 is a plan view of a linearly-arranged substrate for use inthe system of FIG. 1.

[0021]FIG. 3 is a plan view of a two-dimensional substrate for use inthe system of FIG. 1.

[0022]FIG. 4 is a plan view of a pipette tip having a substrate for usein the system of FIG. 1.

[0023]FIG. 5 is a plan view of a plunger-type pipette tip having asubstrate for use in the system of FIG. 1.

[0024]FIG. 6 is a front view of a pipette adapter with bracket for usein the system of FIG. 1.

[0025]FIG. 7 is a side view of the pipette adapter of FIG. 6.

[0026]FIG. 8 is a perspective view of a pipette adapter, supporting asubstrate for use in the system of FIG. 1.

[0027]FIG. 9 is an end view of the pipette adapter of FIG. 8, showingdetails of the supported substrate.

[0028]FIG. 10 is a plan view of a pipette adapter positioned within thesample well of a microtiter plate.

[0029]FIG. 11 is a front view of a flat bottom pipette adapter for usein the system of FIG. 1.

[0030]FIG. 12 is a perspective view of the flat bottom pipette adapterof FIG. 11, showing a substrate at the base of the adapter.

[0031]FIG. 13 is an end view of a flat bottom pipette adapter of FIG.11, showing details of the substrate.

[0032]FIG. 14 is a perspective view of the elements of an opticaldetection station for use in the system of FIG. 1.

[0033]FIG. 15 is a plan view of an alternate embodiment of the detectionstation for use in the system of FIG. 1.

[0034]FIG. 16 presents yet another alternate embodiment of the detectionstation for use in the system of FIG. 1.

[0035]FIG. 17 is a perspective view of a device which may be utilizedfor biopolymer synthesis on a substrate to create a substrate in accordwith the present invention.

[0036]FIG. 18 presents a cross sectional view of the device of FIG. 17.

[0037]FIG. 19 is a plan view of a substrate and backing plate forbiopolymer synthesis, showing a one-dimensional biopolymer array for asubstrate in accord with the present invention.

[0038]FIG. 20 is a plan view of a substrate and backing plate, showingtwo-dimensional biopolymer synthesis for a substrate in accord with thepresent invention.

[0039]FIG. 21 is a perspective view of a jet head-type reagentdeposition apparatus for creating a substrate in accord with the presentinvention.

[0040]FIG. 22 is a plan view of the internal elements of a jet head ofFIG. 21.

[0041]FIG. 23 illustrates a method of affixing a substrate to amanipulator of the present invention.

[0042]FIG. 24 illustrates a first alternative method of affixing asubstrate to a manipulator of the present invention.

[0043]FIG. 25 illustrates a second alternative method of affixing asubstrate to a manipulator of the present invention.

[0044]FIG. 26 is a plan perspective view of a version of the instrumentof FIG. 1, modified to include a jet-head substrate treating apparatus.

BEST MODE FOR CARRYING OUT THE INVENTION

[0045] With reference to FIG. 1, a system 10 utilizing a movablebioarray is shown. Specifically, a robotic arm 12 carries a holder 20which fits and transports bioarray 11, first to the sample, which may bein well 17 of microtiter plate 15 or in vial 16 of rack 14. Although arobotic arm is one form of manipulator which may be used, other simplermanipulators may be employed, such as mechanical movements. A preferredtype of manipulator device is the Biomek, a trademark for an instrumentof Beckman Instruments, Inc. As will be seen below, the substrateportion of bioarray 11 is mounted in a holder having a support regionwhich may be quite small. After the bioarray and the sample have had asufficient incubation or reaction time for interaction of reactants onthe substrate and any target biomolecules which may be present withinthe sample, the robotic arm 12 moves the substrate 11 to the detectionassembly 18 of instrument 10.

[0046] In FIG. 1, optical detection station 18 is presented in a cutawayview showing laser 19, light collector 21, and detector 22. Both thedetection station 18 and the robotic arm 12 may be attached to acomputer, not shown, which generates commands for movement of therobotic arm and receives signals from the detection assembly which may,in turn, be analyzed to determine whether a specific target biomoleculeis present and which may be displayed. The substrate itself is heldwithin a holder 20 which may be coupled to the cantilevered robotic arm12 via pick-up shaft 23 or by some other coupling method. Tracks in thetower 13, the arm 12, and base 59 of the instrument 10, as well ascontrols 24 within the robotic arm 12, position the pick-up unit 25relative to the samples and the detection assembly 18 with x,y,z motion,i.e. three degrees of freedom. A wide range of motion is available overa base the size of a desktop. Many sample wells may be reached as wellas many substrate holders having treated substrates or untreatedsubstrates which may be treated by motion to a nearby location wherereactants may be sprayed or otherwise applied to the substrate.

[0047]FIG. 2 presents a linearly-arranged, flat substrate as a supportportion of a bioarray, with active sites 30 forming bands alongsubstrate 28 which is shaped as a strip. Spaces 26 are provided to placethe plurality of active sites 30 in a spaced-apart relation alongsubstrate 28. In FIG. 3, an area-wide treated substrate is created bypositioning the active sites in a two-dimensional relation on a planarsheet substrate. Active sites 30 are positioned in spaced-apart relationon substrate 28, as before. As illustrated in FIGS. 2 and 3, the activesites 30 may be bands, as in FIG. 2, or spots, as in FIG. 3, or someother shape. The spots are in known locations and are specific for atarget biomolecule. A plurality of linear substrates of FIG. 2 may alsobe arranged in parallel to create a two-dimensional bioarray. The sizeof the substrate is typically a few centimeters on a side, but could besmaller or larger.

[0048] By way of example, the reactants forming the active sites maycomprise complementary DNA strands for detection by DNA hybridization orthey may comprise immunological biomolecules for detection byimmunological complexing, such as formation of antigen-antibodycomplexes.

[0049] The device of FIG. 4 represents one example of a substrate holderwhich may be used to present the bioarray to the sample. A pipette tip27 is shown with a substrate 11 supported longitudinally along an insidewall. The substrate is preferably positioned along an inside wall of thepipette tip and the pipette tip is comprised of an optical glass orplastic allowing optical inspection of the substrate while the substrateis positioned inside the pipette tip. The sample is drawn into thepipette tip by aspiration and allowed to interact with substrate 11.Pipette tip 27 preferably has at least one flat surface, i.e. thesurface opposite the substrate, for accurate optical inspections. Thisfeature and a narrow bore also help minimize the amount of samplenecessary and place the sample and substrate in close proximity. Thepipette tip 27 may be used in conjunction with a rubber bulb, vacuumpump, robotic pipettor as in FIG. 1, or other device. The term “pipettetip” is meant to include pipettes, such as long cylindrical glass orplastic pipettes which are designed to operate in connection with asimple suction device.

[0050]FIG. 5 shows a second embodiment of a substrate holder wherein thesubstrate 11 is housed within a plunger-type pipette tip, in the samemanner as the FIG. 4 embodiment. Pipette tip 29 also has a narrow boreand flattened surface. The sample is drawn into the plunger-type pipettetip 29 through withdrawal of plunger 31. Positive displacement of thesample is used to eject the sample from the pipette, as by depression ofplunger 31 or by some other fluid manipulation. The embodiment of FIG. 5has an advantage over the embodiment of FIG. 4 in that the sample doesnot drain from the pipette tip when the pipette tip of FIG. 5 isdetached from a pipetting tool.

[0051] In FIGS. 6-9, a substrate holder takes the form of a pipetteadapter 32 characterized by a bracket 33 at one end. The bracket hasopposed prongs 36 a-b, easily visible in FIG. 8, which support the endsof the substrate which is part of the bioarray. The opposite end of thebracketed pipette adapter preferably has a coupler 37 for joining arobotic or standard pipetting tool.

[0052] In FIG. 8, the coupler 37 is depicted as a hollow cone which mayfit the conical shaft of a robotic or standard pipetting tool with anappropriate securing mechanism, such as a friction fit, with provisionfor ejection of the adapter for use. Many different types of couplersmay be used, however. Of course, the pipette adapter 32 need not haveany coupler, but it is preferred that the adapter have a gripper orother means for manipulating the adapter so, for example, the adaptermay be moved into and out of sample wells easily.

[0053] The bioarray is preferably oriented so that the active sites facedownward. Thus, when the adapter is placed within a sample well, as inFIG. 10, contact of the active sites of the substrate and the sample isfacilitated. Pipette adapter 32 is preferably equipped with knobs 35 onthe prongs of the bracket, visible in FIGS. 6-9. These knobs positionthe substrate slightly above the bottom of the sample well, and protectthe treated substrate from physical abrasion and contamination. Withoutsuch knobs, placement of the adapter into the well may press thesubstrate so close to the bottom of the well as to exclude sample fromthe face of the substrate, preventing proper contact and interaction ofthe sample and the substrate. Bracketed pipette adapter 32 alsopreferably contains a ring or disk-shaped evaporation barrier 34 whichis disposed about the midsection of the adapter, or at the base of thebracket portion. Because some samples may easily evaporate, evaporationbarrier 34 is preferably provided to protect the sample during thesubstrate and sample incubation period. In FIG. 10, evaporation ring 34is seen providing a barrier when pipette adapter 32 is inserted intowell 17, thus limiting the exposure of sample 38. The diameter ofpipette adapter 32, and particularly bracket 33, is sufficiently narrowin order to easily fit within the microtiter plate's well, as also seenin FIG. 10.

[0054]FIG. 9 presents an end view of the bracketed pipette adapter 32.This view is indicated by axis 9-9 of FIG. 6, viewed in the direction ofthe arrows. From the end view, the substrate 11 is more clearly visiblein its preferred downward facing orientation. The substrate of FIG. 9 isin a linearly arranged, but segmented, form. Thus strips 11 a, 11 b, and11 c are positioned in a generally parallel arrangement and secured byprongs 36 a-b of the bracket 33. The nature and shape of the substratemay be easily adapted to sample, applicator, and space considerations.The two-dimensional substrate of FIG. 3 would adapt easily to bracketedpipette adapter 32. The substrate is held by prongs 36 a-b by adhesion,welding, clamping, or any other means for gripping which will notinterfere with the testing of the sample. In the end view of FIG. 9,evaporation disk 34 is visible beyond the prongs and the substrate.

[0055] In FIGS. 11-13, a flat-bottom pipette adapter 39 is utilized tosupport and transport the bioarray. Flat-bottom pipette adapter 39 has aflat bottom surface 40, visible in FIG. 12, and may have a coupler 37 atan end opposite to the flat bottom surface 40 for fitting the adapter topipetting tools or a robotic arm, as in the bracketed pipette adapterembodiment. Similarly, flat bottom pipette adapter 39 may have a simplemeans for manually gripping the pipette adapter and applying thesubstrate 11 to the sample. Also, as with the bracketed pipette adapter32, the flat bottom adapter 39 is preferably outfitted with knobs 35 andevaporation ring 34. In FIG. 13, the substrate 11 is seen to be atwo-dimensional array of spots or dots, as in FIG. 3. Evaporation ring34 is visible in this view, taken along axis 13-13 of FIG. 11, andsituated beyond flat bottom surface 40.

[0056] In FIG. 14, details of the internal elements of a biomoleculeprobe station are shown. Although various probe methods are available,the optical detection station 18 is an example. The station may be partof an analysis machine having a robotic arm, such as that shown inFIG. 1. The robotic arm, pipetting tool, or other substrate holderpositions the substrate, after it has interacted with the sample, in thepath of a laser beam. Laser 41 creates a beam which impinges upon theactive sites of the substrate 11, which is held within bracketed pipetteadapter 32. The wavelength of the beam is selected to cause the returnof a radiation signature from target molecules bound to the substrate.Such a signature comes from an optically detectable characteristicradiation pattern of the bound target molecules when excited byradiation of the beam, such as a characteristic band of fluorescentwavelengths. Time gated fluorescence, or other optical signalenhancement techniques, may optionally be used. The incident beam fromthe laser is scanned across the active sites of the treated substrate byrelative motion of the substrate and the beam. Light emitted from theactive sites is collected by light collector 42 and directed to detector43, which may be a photomultiplier tube, CCD array, or other detectiondevice, and which is preferably associated with a computer for anyfurther analysis or display of the signals received from the substrate.Additional optical elements, such as wavelength selective filters, maybe disposed in either the incident beam or the return light, as requiredby the characteristic radiation signature. Scanning may be accomplishedby moving the substrate relative to the laser beam, by utilizing ascanning reflector such as a galvo mirror or polygonal mirror, or bysome other well known means. Alternatively, the area of the laser beammay be expanded such that the entire area of the array is illuminatedsimultaneously, and scanning is not required.

[0057] The bioarray is optically probed by the beam for determining theextent of complexing of the reactants in the active sites of thesubstrate with target biomolecules in the sample. The optical inspectionmay be for fluorescent signals, reflectance, absorbance, lightscattering, or chemiluminescence, among others. Details of the opticalsystem may vary according to the nature of the signal to be detected.FIG. 14 illustrates a substrate 11 within bracketed pipette adapter 32and facing in a downward orientation for impingement by the laser beam.This arrangement of the elements of the detection assembly is presentedas an example of the arrangement of the detection assembly 18 of FIG. 1.In either case, the robotic arm may easily move the substrate and theassociated bracketed pipette adapter to the detection assembly after theappropriate sample incubation period. The robotic arm may, however, becapable of moving the substrate so that it is oriented vertically, or insome other manner, relative to the laser beam. Also, the laser sourcefor the excitation path may be positioned in a manner other than shownin order to impinge upon the substrate. Optical fibers may be employedto direct the beam or the return signal.

[0058]FIG. 15 presents a detection arrangement for probing of asubstrate within a pipette tip. Pipette tip 27 has an optical surface soas not to interfere with the optical inspection. As with the bracketedpipette adapter, laser 41 impinges upon the substrate 11 and emittedsignals from the substrate are gathered by light collector 42 anddirected to detector 43 where the signals may be sent onto a computerfor further analysis. The excitation beam from the laser impinges on thesubstrate through the wall of the pipette tip.

[0059] Arrow A of FIG. 15 indicates one example of how the substrate maybe scanned, i.e. by providing a vertical motion to the pipette tip, viathe robotic arm or some other mechanism. In FIG. 16, the substrate 11within pipette tip 27 is scanned by the incident laser beam through theaction of a scanning reflector 44, which may be rotated in direction Bto cause scanning of the substrate 11. An automated apparatus such asinstrument 10 of FIG. 1 may have a plurality of detection assemblies towhich the robotic arm may move the substrate for reading, depending uponthe type of manipulator used for the substrate, the type of signals tobe read from the substrate, and the nature of the substrate and targetbiomolecules thereon.

[0060] The substrate may be formed by the device shown in FIGS. 17-20,which allows biopolymer synthesis on a solid support. The substrate maybe used directly, or the biopolymers created by the device may becleaved from the substrate and affixed to another substrate in anydesired format. The method and apparatus depicted in FIGS. 17-20 are thesubject of commonly-assigned U.S. Pat. No. 5,429,807, which isincorporated herein by reference. The device of FIG. 17 presents asynthesis device 45 which is a thick block having a plate surface 46within which are a plurality of grooves or channels 50. Channels 50 areconnected to tubing 47 at the underside of block 45 for flowing reagentsthrough channels 50. The cross sectional view of FIG. 18, taken alongaxis 18-18 of FIG. 17, more clearly illustrates the reagent flow throughthe block wherein tubing 47 a is connected to an inlet tubing connector49 which communicates with channel 50. Tubing 47 b communicates withoutlet tubing connector 52 which in turn communicates with channel 50.Thus, reagents may be caused to flow through any of the channels 50.

[0061] To perform a synthesis, a solid support material, such as a sheetof activated polypropylene, may be placed on top of the channels of theblock. A backing plate may be used to sandwich the polypropylenesubstrate, allowing the flexible polypropylene to seal against thechannels 50 of the block 45. The backing plate 52 of FIGS. 19 and 20 mayhave holes 53 which may be aligned with holes 48 of block 45. Thebacking plate and the block may then be secured to one another.Synthesis or biopolymerization may be performed by activating thesurface of the substrate, if necessary, and by flowing reagents throughthe channels, to cause formation of strands of biopolymers anchored tothe substrate. This results in a one-dimensional array of biopolymers54, as seen in FIG. 19. If desired, the block may be repositioned withrespect to the substrate 55 and the process repeated. In FIG. 20,one-dimensional arrays 54 a-b are presented in 90° offset orientations.The areas of overlap 56 provide new biopolymers having elements of eachone-dimensional array 54 a-b. Indexing pins 58, visible in FIGS. 19 and20, may be utilized to position the substrate 55 in relation to theapplicator. Indexing pins 58 mate with holes 57 in block 45. Theresulting arrays may be utilized as is, or may be cleaved from thepolypropylene substrate 55 and affixed to some other support.Additionally, the substrate 55 having the arrays may be segmented andattached to substrate 28, in the manner of FIGS. 8 and 9. Althoughchannels 50 are illustrated, cavities for reagent flow having some othershape may be used. Although polypropylene is presented in the abovediscussion, other substrates such as glass, Pyrex, silicon, polystyrene,etc. may be utilized as supports for synthesis, as suggested in PCTapplication No. WO 93/09668.

[0062] With reference to FIG. 21, another method of creating a substrateis shown, utilizing movable nozzles. A support 28 is positioned onsupport frame 65 within a spray station. A plurality of ink jet-typeheads 60 with nozzles 61 at the spray station are used to selectivelydeposit reactants on support 28 to create the plurality of active sites.Such heads are well-known in the field of ink jet printing. In thepresent invention, such heads are adapted for dispensing the reactantsonto the desired locations of the substrate. If necessary, the substratemay be activated to receive and immobilize the reactants.

[0063] In FIG. 21, a plurality of different jet heads 60, each having anozzle 61, may be moved on respective rails 62 in the directionindicated by arrow C. Each of the jet heads 60 dispenses a differentreactant. Motion control of the jet heads 60 along rails 62 may beprovided by a computer. The array may be moved by an actuator 63, whichcauses an arm 64 to move support frame 65 carrying the support 28. Theactuator 63 may be a linear motor similar to that used to move magneticheads in disc drives. In the situation where the substrate for thetreated substrate is a strip, fixed position jet heads may be desired.

[0064]FIG. 22 illustrates a typical ink jet-type dispensing head asapplied in this invention. Reactant contained in reservoir 67 passesthrough supply tube 69 to piezoelectric pumping chamber 66, and throughchamber 66 to nozzle 69. Electrical pulses applied to pumping chamber 66cause it to expand and contract in volume. Each time a pulse is appliedand removed, this expansion and contraction event ejects a droplet 70 ofreactant from the nozzle. Additional details of the design and operationof such a reactant dispensing device are disclosed in previouslyreferenced U.S. Pat. No. 4,877,745. In addition to dispensing reactants,such a dispensing head might be employed to dispense ink or dye onto thesubstrate to form barcode patterns for machine reading of theidentification of a bioarray.

[0065] Another method of preparing the bioarray is by a techniqueanalogous to a printing method. In this technique an analyte isdeposited on a substrate by stamping or embossing a very thin layer withan array of analyte spots at desired locations. For example, an antigenattached to a molecule anchored to the substrate by pressure contactwill combine with an appropriate antibody associated with a specifictarget biomolecule. The antibody may be fluorescent for opticaldetection.

[0066] Other methods of preparing the substrate may be used,particularly photolithographic techniques. In a journal article entitled“Light-Directed, Spatially Addressable Parallel Chemical Synthesis” byS. Fodor et al. in Science, Feb. 15, 1991, p. 767, the authors describea method of synthesizing complex compounds at spatially discretelocations defined by photomasks of the type used in the semiconductorindustry. Molecular building blocks are deposited at desired locationsby exposing underlying building blocks, i.e. “deprotecting” theunderlying block for a reaction with the superposed building block.Successive building blocks are added until a desired compound is formed.The location of each compound is precisely known from the mask set andthe sites may be very closely spaced, limited only by the diffraction oflight.

[0067] A method of imaging, i.e. probing, a substrate having microscopicfeatures is by means of condensation figures (CFs) described in ajournal article entitled “Imaging of Features on Surfaces byCondensation Figures” by G. Lopez et al. in Science, Apr. 30, 1993, p.647. The authors describe the formation of an array of tiny droplets ona cold surface having an array of spots which are not wet by thedroplets. The spots could be the complex compounds described in thepreceding paragraph. The droplets are observed with microscope optics.

[0068] Still another bioarray forming technique is described in anarticle by B. Healey et al. in Science, Aug. 25, 1995 p. 1078. Theauthors deposited microscopic polymer arrays on a flat substrate bydepositing a layer of polymerization solution on a flat plate, such as aglass chip which had been activated for adhesion with the solution. Abundle of fibers was brought into contact with the solution and thenbacked off and the substrate rinsed. Light was directed into thenon-contacted end of the fiber bundle to cause polymer deposition on thesubstrate below the fibers of the fiber bundle. Polymer spots of a 2.0micrometer diameter and a spacing of 4.0 micrometers were produced.

[0069] Yet another bioarray forming technique is the Southern blottingmethod in which hybridization is used simultaneously on a large numberof DNA segments. DNA is fragmented, electrophoresed, denatured andtransferred from a gel to filter paper. Positions of numerous fragmentsare established. The DNA fragments are robotically moved in accord withthe present invention and combined with radioactive phosphorous labelledRNA which can be identified. The degree of DNA-RNA complementation, i.e.probing of the sample, can be determined by autoradiography.

[0070] In another bioarray forming technique a polyunsaturatedpolymerized lipid layer is applied to a support. The lipids have amember of a specific binding pair bound to one end. The lipids have anoptical characteristic which is modified upon complexing the othermember of the binding pair. Such an optical characteristic can bepolarization of light and such light is used to probe the bioarray.

[0071] In FIG. 23, affixing of bioarray 11 to the flat bottom surface 40of flat bottom pipette adapter 39, seen in FIGS. 10-12, is given as anexample. Specifically, in FIG. 23, a two-dimensional bioarray 11 ispreformed and then attached to the flat bottom surface 40 of pipetteadapter 39. FIG. 24 presents an alternative wherein the substrate 28 isaffixed to flat bottom surface 40 of the pipette adapter, beforereactants are caused to become immobilized on the substrate to form thebioarray 11. This alternative works particularly well with the printingtype of creation for substrates as discussed with reference to FIGS. 21and 22. In FIG. 25, it can be seen that the bare substrate 28 may be anintegral part of the adapter as fabricated, for instance by injectionmolding. The substrate 28 is then activated, indicated by shading inFIG. 25, and then the reactants are deposited or otherwise caused toattach to substrate 28.

[0072] Although the method of the present invention is designed fordetection of target biomolecules in a sample, quantification of thetarget biomolecules is possible by, for example, recording the samplevolume exposed to the substrate, quantifying the degree ofcomplementation at the active sites of the substrate, and calculatingthe amount of target biomolecule present from these two values.Quantification of the degree of complementation may be performed, e.g.,by measuring the percentage of active sites which arefluorescently-labeled or give some other optical signal indicatingcomplementation. Additionally, affixing an excess amount of reactants tothe substrate compared to the amount of suspected target biomolecules ofthe sample is a preferred practice and makes quantification moreaccurate.

[0073] Referring to FIG. 26, an alternate embodiment of instrument 10 ofFIG. 1, incorporating the jethead-type reagent dispensing means, isshown. As before, the instrument contains a detection assembly 18 andhas a location for placement of the samples. In this case, the samplesare within wells 17 of microtiter plate 15. Additionally, FIG. 26 showsa location for a holder 20 for a substrate. For simplicity, pipetteadapter 32 has a bioarray held within opposed prongs of a bracket, asillustrated in FIGS. 6-9. Specifically, pipette adapter rack 81 is shownhaving a plurality of pipette adapters 32 a. Here, the instrument alsocontains a jethead dispensing device 80. The dispensing device is of thetype discussed with regard to FIGS. 21 and 22. As with the instrument ofFIG. 1, this variation has a robotic arm 12 attached to a tower 13which, in turn, is attached to a base 59. As before, tower 13 and base59 have tracks for providing both vertical and horizontal motion to therobotic arm. Additionally, in FIG. 26, the detection assembly 18 anddispensing device 80 are depicted as blocks having holes. These blocksillustrate that the instrument is provided with various stations, eachhaving dedicated operations. The holes enable access to the holder whichis attached to the robotic arm.

[0074] In operation, the instrument of FIG. 26 provides motion to thebioarray via the robotic arm which picks up a pipette adapter having asubstrate support from rack 81, and then moves the pipette adapter intodispensing device 80. Within dispensing device 80, the support may beactivated, if necessary, and dispensing or printing of reactants uponthe support to create a substrate occurs. Then, robotic arm 12 moves thepipette adapter from dispensing device 80 into contact with a sample, asby placing pipette adapter 32 within a sample well 17 of *microtiterplate 15. The pipette adapters 32 b shown in the microtiter plate 15have been deposited into the wells by robotic arm 12 for interaction ofthe substrates and the samples. After the appropriate incubation time,robotic arm 12 picks up each pipette adapter and moves it to detectionassembly 18 for detection, as before.

[0075] In the above description, the robotic arm moved the pipetteadapter, with holder and bioarray, to a sample location, such as amicrotiter plate. However, the robotic arm could pick up sample in apipettor and bring it to a stationary holder where the pipettor coulddispense the sample onto the holder. Then, the same robotic arm, oranother one, with an appropriate gripper could move the holder to adetection station.

[0076] The detection station could be any of the optical types describedabove, but could also be a radioactive tag detector if the immobilizedreactants for the target biomolecule had been radioactive. Also, if thetag was a moiety suitable for detection by laser desorption massspectrometry (LD-MS), then an LD-MS detection system could be used.Other tags and detection systems will be evident to those skilled in theart.

1. A system for detecting the presence of a target biomolecule within asample comprising: a support surface treated with distinct reactantsimmobilized thereon in a spaced-apart relation, forming a substratehaving a plurality of spaced-apart active sites, at least one of thereactants being reactive with a target biomolecule to form a boundcomplex having a detectable characteristic, holder means for supportingthe substrate, an inspection station having means for probing thespaced-apart active sites of the substrate for said detectablecharacteristic of the sample, and manipulator means for bringing theholder means into contact with the sample and into said inspectionstation.
 2. The system of claim 1 further comprising a plurality ofsample reservoirs arranged in fixed, closely spaced, known locations,said manipulator means comprising a robotic arm movable among saidsample holders and said inspection station.
 3. The system of claim 1wherein the holder means comprises a pipettor.
 4. The system of claim 2wherein the robotic arm comprises a movable upright tower supporting across beam, wherein said holder means receives cantilever support fromthe cross beam.
 5. The system of claim 1 wherein the substrate hasopposed ends fixed to the holder means and an unsupported region betweenthe opposed ends.
 6. The system of claim 5 wherein the holder meanscomprises a pipette tip within which the sample may be drawn byaspiration.
 7. The system of claim 6 wherein the pipette tip supportsthe substrate longitudinally along an inside wall of the pipette tip. 8.The system of claim 6 wherein the pipette tip has an optical surface. 9.The system of claim 6 wherein the pipette tip has a narrow bore forminimizing sample volume and maximizing proximity of the sample and thesubstrate.
 10. The system of claim 6 wherein the pipette tip has atleast one flattened surface for minimizing sample volume and maximizingproximity of the sample and the substrate, and for enhancing opticalinspection of the substrate.
 11. The system of claim 5 wherein theholder means comprises a plunger-type pipette tip within which thesample may be drawn by withdrawal of the plunger and resultingaspiration of the sample and the sample may be dispensed by depressionof the plunger.
 12. The system of claim 11 wherein the plunger-typepipette tip supports the substrate longitudinally along an inside wallof the plunger-type pipette tip.
 13. The system of claim 11 wherein theplunger-type pipette tip has an optical surface.
 14. The system of claim11 wherein the plunger-type pipette tip has a narrow bore for minimizingsample volume and maximizing proximity of the sample and the substrate.15. The system of claim 1 wherein the holder means comprises a pipetteadapter having a bracket for holding the substrate.
 16. The system ofclaim 15 wherein the pipette adapter has a coupler for fitting thepipette adapter to a pipetting tool.
 17. The system of claim 15 whereinthe pipette adapter is sufficiently narrow at the bracket to immerse thebioarray in a sample well of a microtiter plate.
 18. The system of claim15 wherein the bracket of the pipette adapter holds the substratebetween opposed prongs of the bracket.
 19. The system of claim 15wherein the substrate is planar and the pipette adapter supports thesubstrate in an orientation such that the active sites face downward.20. The system of claim 15 further comprising knobs on the bracketprongs, the knobs positioning the substrate a fixed distance from thesample in a position to protect the substrate from abrasion andcontamination.
 21. The system of claim 15 further comprising anevaporation barrier disposed about the pipette adapter.
 22. The systemof claim 1 wherein the manipulator means comprises a pipette adapterhaving a flat bottom surface for bearing the substrate.
 23. The systemof claim 22 wherein the pipette adapter has a coupler means for fittingthe pipette adapter to a pipetting tool.
 24. The system of claim 22wherein the pipette adapter is sufficiently narrow at the flat bottomsurface to immerse the substrate in a sample well of a microtiter plate.25. The system of claim 22 wherein the substrate is attached to the flatbottom surface.
 26. The system of claim 22 wherein the substrate isplanar and the pipette adapter supports the substrate in an orientationsuch that the active sites face downward.
 27. The system of claim 22further comprising knobs on the flat bottom surface of the pipetteadapter, the knobs positioning the substrate a fixed distance from thesample in a position to protect the substrate from abrasion andcontamination.
 28. The system of claim 22 further comprising anevaporation barrier disposed about the flat-bottom pipette adapter. 29.The system of claim 1 wherein the substrate is a flat bottom surface ofa pipette adapter.
 30. The system of claim 1 wherein the substrate has alinear arrangement of the reactants.
 31. The system of claim 30 whereinthe substrate has a segmented linear arrangement of the reactants. 32.The system of claim 1 wherein the substrate has an area-wide arrangementof the reactants.
 33. The system of claim 1 wherein the substrate ispreformed with reactants prior to being affixed to the manipulatormeans.
 34. The system of claim 1 wherein the substrate is affixed to themanipulator means before reactants are immobilized on the substrate. 35.The system of claim 1 wherein the manipulator means has a surface andthe substrate is formed directly on the manipulator means by utilizingthe surface as the substrate.
 36. The system of claim 1 wherein thereactants comprise complementary DNA strands.
 37. The system of claim 1wherein the reactants comprise immunological biomolecules.
 38. Thesystem of claim 1 wherein the substrate comprises a polypropylenemember.
 39. The system of claim 1 wherein the substrate comprises aglass member.
 40. The system of claim 1 wherein the reactants areanchored on the substrate via biopolymer synthesis.
 41. The system ofclaim 1 wherein the reactants are anchored on a solid support member viabiopolymer synthesis with subsequent segmenting of the solid supportmember and mounting of the solid support member on the support surfaceto form said substrate.
 42. The system of claim 1 wherein the substratehas reactants deposited at discrete known locations on the substrate.43. The system of claim 42 wherein reactants are dispensed onto thesubstrate through movable nozzles.
 44. The system of claim 1 wherein thereactants are affixed to the substrate via covalent linkage.
 45. Thesystem of claim 1 wherein the reactants are affixed to the substrate viaphysical absorption.
 46. The system of claim 1 wherein the inspectionstation further comprises: means for impinging the light beam on thesubstrate, a light collector for collecting optical signals emitted fromthe substrate, and a detector disposed to detect the collected signalsfrom the light collector.
 47. The system of claim 46 wherein thedetector comprises a fluorescence detector.
 48. The system of claim 1wherein the inspection station comprises a beam passing through thesubstrate to a detector measuring absorbance.
 49. The system of claim 1wherein the inspection station comprises a beam impinging on thesubstrate with a detector disposed for measuring reflectance.
 50. Thesystem of claim 1 wherein the inspection station comprises a beamimpinging on the substrate with a detector disposed for measuring lightscattering.
 51. The system of claim 1 wherein the inspection stationcomprises a beam impinging on the substrate with a detector disposed formeasuring chemiluminescence.
 52. A method for detecting the presence ofa target biomolecule within a sample, the method comprising: (a)immobilizing distinct reactants in a spaced-apart relation on a surfaceof a support member, thereby forming a substrate having a plurality ofactive sites, at least one of the reactants being reactive with a targetbiomolecule to form a bound substance having a detectablecharacteristic, (b) affixing the substrate onto a holder, (c) moving theholder into contact with the sample for a period of time, (d) removingunbound biomolecules from the substrate, and (e) moving the substratesto a location for probing the active sites of the substrate, and (f)detecting a detectable characteristic of the bound structure on thesubstrate.
 53. The method of claim 52 further defined by roboticallymoving the holder.
 54. The method of claim 52 wherein immobilizing thereactants comprises synthesizing reactants by biopolymerizationreactions anchored at discrete locations on the substrate.
 55. Themethod of claim 52 wherein immobilizing the reactants further comprisessynthesizing reactants by biopolymerization reactions anchored atdiscrete locations on a solid support surface, segmenting the solidsupport surface to separate distinct reactants, and mounting thesegmented solid support surface to the support member to form saidsubstrate with spaced-apart active sites.
 56. The method of claim 52wherein immobilizing the reactants further comprises dispensing thereactants at discrete locations on the substrate via movable nozzles.57. The method of claim 56 further comprising activating the surface ofthe support member before dispensing the reactants.
 58. The method ofclaim 56 further comprising affixing an optical bar code pattern to thesupport member for identification of the substrate.
 59. The method ofclaim 56 wherein dispensing the reactants at discrete locations furthercomprises directing the reactants to form round spots on the supportmember.
 60. The method of claim 56 wherein dispensing the reactants atdiscrete locations further comprises directing the reactants to formlinear bands on the support member.
 61. The method of claim 56 whereindispensing the reactants at discrete locations further comprisesdeflecting the reactants to form a desired pattern on the supportmember.
 62. The method of claim 52 wherein immobilizing the reactantsfurther comprises affixing excess amount of reactants to the supportmember compared to the amount of suspected target biomolecules of thesample which would react with the reactants.
 63. The method of claim 52wherein immobilizing the reactants comprises forming covalent linkagesof the reactants with the support member.
 64. The method of claim 52wherein immobilizing the reactants comprises adsorption of the reactantson the support member.
 65. The method of claim 52 wherein affixing thesubstrate onto a holder further comprises affixing the substrate in alinear arrangement along an inside wall of a pipette tip, and whereincontacting the sample within the substrate further comprises aspiratingthe sample into the pipette tip.
 66. The method of claim 52 whereinaffixing the substrate onto a holder further comprises affixing thesubstrate in a linear arrangement along an inside wall of a plunger-typepipette tip, wherein contacting the sample with the substrate furthercomprises drawing the sample into a plunger-type pipette tip bywithdrawal of the plunger, and wherein removal of the substrate from thesample further comprises dispensing the sample from the plunger-typepipette tip by depression of the plunger.
 67. The method of claim 52wherein affixing the substrate onto a holder further comprises affixingthe substrate between opposed prongs of a bracket of a pipette adapter,and wherein contacting the sample with the substrate further comprisesplacing the sample in a vial or well and placing the pipette adapter,with the prongs in an inverted position, into the vial or well.
 68. Themethod of claim 52 wherein affixing the substrate onto a holder furthercomprises affixing the substrate to a flat, bottom surface of a pipetteadapter and wherein contacting the sample with the substrate furthercomprises placing the sample in a vial or well and placing the pipetteadapter, with the flat, bottom surface down, into the vial or well. 69.The method of claim 52 further comprising exposing the substrate to adevelopment chemical to enhance visualization of complementation, afterremoving the substrate from the sample and before probing the activesite.
 70. The method of claim 69 wherein exposing the substrate to adevelopment chemical further comprises exposing the substrate to afluorescence dye.
 71. The method of claim 52 wherein detecting adetectable characteristic comprises inspecting the bound structure witha light beam for fluorescence.
 72. The method of claim 52 whereindetecting a detectable characteristic comprises inspecting the boundstructure with a light beam for absorbance.
 73. The method of claim 52wherein detecting a detectable characteristic comprises inspecting thebound structure with a light beam for reflectance.
 74. The method ofclaim 52 wherein detecting a detectable characteristic comprisesinspecting the bound structure with a light beam for light scattering.75. The method of claim 52 wherein detecting a detectable characteristiccomprises inspecting the bound structure for chemiluminescence.
 76. Themethod of claim 52 further comprising recording the sample volumeexposed to the substrate, quantifying the amount of bound substance atthe active sites on the substrate, and calculating the amount of thetarget biomolecule present in the sample from the sample volume exposedto the substrate and the amount of bound substance at the active sitesof the substrate.
 77. A holder for bearing a substrate having reactantbinding agents for at least one target biomolecule in a sample to form abound substance having a detectable characteristic for detection oftarget biomolecules within the sample, the holder comprising: a pipettetip for use in conjunction with a pipettor, the pipette tip bearing asubstrate with distinct reactants in a spaced-apart relation, thesubstrate disposed longitudinally along an inside wall of the pipettetip, wherein the pipette tip has means for aspirating the sample intothe pipette tip for contact between the reactants and the sample on thesubstrate.
 78. The holder of claim 77 wherein the aspirating means is arobotic pipettor.
 79. The holder of claim 77 wherein the aspiratingmeans is a hand-held pipettor.
 80. The holder of claim 77 wherein thepipette tip has a narrow bore to maximize proximity of the substrate andthe sample.
 81. The holder of claim 77 wherein the pipette tip has atleast one flattened surface.
 82. The holder of claim 77 wherein thepipette tip has an optical surface opposite the substrate.
 83. A holderfor bearing a substrate having reactant binding agents for at least onetarget biomolecule in a sample to form a bound substance having adetectable characteristic for detection of target biomolecules withinthe sample, the holder comprising a pipette tip having a plunger andbearing a substrate with distinct reactants in a spaced-apart relation,the substrate disposed longitudinally along an inside wall of thepipette tip, wherein the plunger draws the sample into the pipette tipfor contact between the reactants and the sample on the substrate. 84.The holder of claim 83 wherein withdrawal of the plunger is automated.85. The holder of claim 83 wherein the pipette tip has a narrow bore tomaximize proximity of the substrate and the sample.
 86. The holder ofclaim 83 wherein the pipette tip has at least one flattened surface. 87.The holder of claim 83 wherein the pipette tip has an optical surfaceopposite the substrate.
 88. A holder for bearing a substrate havingreactant binding agents for at least one target biomolecule in a sampleto form a bound substance having a detectable characteristic fordetection of target biomolecules within the sample, the holdercomprising: a pipette adapter having a bracket with opposed prongs, theopposed prongs bearing the substrate with distinct reactants in aspaced-apart relation, the substrate disposed in a downward-facingorientation, wherein the bracket of the pipette adapter is placed on thesample for contact of the sample with the substrate.
 89. The holder ofclaim 88 wherein the pipette adapter has a coupling means for couplingthe pipette adapter to a robotic arm.
 90. The holder of claim 88 whereinthe pipette adapter has a coupling means for coupling the pipetteadapter to a hand-held pipette.
 91. The holder of claim 88 wherein thebracket of the pipette adapter is sufficiently narrow in diameter to fitwithin a sample well of a microtiter plate.
 92. The holder of claim 88wherein the bracket prongs of the pipette adapter have knobs positioningthe substrate a fixed distance from the sample.
 93. The holder of claim88 wherein the pipette adapter has an evaporation barrier disposed aboutthe pipette adapter.
 94. A holder for bearing a substrate havingreactant binding agents for at least one target biomolecule in a sampleto form a bound substance having a detectable characteristic fordetection of target biomolecules within the sample, the holdercomprising: a pipette adapter having an end with a flat bottom surface,the flat bottom surface bearing the substrate with distinct reactants ina spaced-apart relation, the substrate disposed in a downward facingorientation, wherein the flat bottom surface of the pipette adapter isplaced on the sample for contact of the sample with the substrate. 95.The holder of claim 94 wherein the pipette adapter has a coupling meansfor coupling the pipette adapter to a robotic arm.
 96. The holder ofclaim 94 wherein the pipette adapter has a coupling means for couplingthe pipette adapter to a hand-held pipettor.
 97. The holder of claim 94wherein the end of the pipette adapter having the flat bottom surface issufficiently narrow in diameter to fit within a sample well of amicrotiter plate.
 98. The holder of claim 94 wherein the flat bottomsurface of the pipette adapter has knobs positioning the substrate afixed distance from the sample.
 99. The holder of claim 94 wherein thepipette adapter has an evaporation barrier disposed about the pipetteadapter.